Instrumentation of spectrofluorimetry

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Detailed information about instrumentation of spectrofluorimetry with pictures

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INSTRUMENTATION OF SPECTROFLUORIMETRY:

INSTRUMENTATION OF SPECTROFLUORIMETRY P.VINOTH KUMAR , M.PHARM,I YEAR, MTPG&RIHS, PUDUCHERRY-6 .

COMPONENTS:

COMPONENTS SOURCE OF LIGHT FILTERS AND MONOCHROMATORS SAMPLE CELLS DETECTORS

AN IDEAL SPECTROFLUORIMETER :

AN IDEAL SPECTROFLUORIMETER The individual components must have the following characteristics: the light source must yield a constant photon output at all wavelengths; the monochromator must pass photons of all wavelengths with equal efficiency; the monochromator efficiency must be independent of polarization; the detector (photomultiplier tube) must detect photons of all wavelengths with equal efficiency. Unfortunately, light sources, monochromators, and detectors with such ideal characteristics are not available . As a result, one is forced to compromise on the selection of components and to correct for the non-ideal response of the instrument.

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Power supply Source primary filter secondary filter Detector Sample cell Slit Data processor

SOURCES OF LIGHT:

SOURCES OF LIGHT Arc and Incandescent Xenon Lamps High-Pressure Mercury Lamps Xenon–Mercury Arc Lamps Quartz–Tungsten Halogen (QTH) Lamps LED Light Sources

High-Pressure Mercury Lamps :

High-Pressure Mercury Lamps In general mercury lamps have higher intensities than Xenon lamps, but the intensity is concentrated in lines. These lamps are only useful if the Hg lines are at suitable wavelengths for excitation.

Arc and Incandescent Xenon Lamps :

Arc and Incandescent Xenon Lamps At present the most versatile light source for a steady-state spectrofluorometer is a high-pressure xenon ( Xe ) arc lamp.These lamps provide a relatively continuous light output from 250 to 700nm , with a number of sharp lines. The gas in xenon lamps is under high pressure (about 10 atmospheres), and explosion is always a danger. A xenon lamp that is on should never be observed directly . The extreme brightness will damage the retina, and the ultraviolet light can damage the cornea.

Xenon–Mercury Arc Lamps :

Xenon–Mercury Arc Lamps These have higher intensities in the ultraviolet than Xenon lamps, and the presence of Xenon tends to broaden the spectral output . When first started the Hg– Xe lamp output is due mostly to Xenon. As the lamp reaches operating temperature all the Hg becomes vaporized, and the mercury output increases .

Quartz–Tungsten Halogen (QTH) Lamps   :

Quartz–Tungsten Halogen (QTH) Lamps   These lamps provide continuous output in the visible and IR regions of the spectrum. there is presently increasing interest in fluorophores absorbing in the red and near infrared (NIR), where the output of a QTH lamp is significant.

LED Light Sources :

LED Light Sources LEDs are just beginning to be used as light sources in spectrofluorimeters. 8 LEDs can be placed close to the samples, and if needed the excitation wavelength can be defined better by the use of an excitation filter. 9 LEDs have the advantage of long life and low power consumption.

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FILTERS AND MONOCHROMATORS :- Filters: these are nothing but optical filters works on the principle of absorption of unwanted light and transmitting the required wavelength of light. In inexpensive instruments fluorimeter primary filter and secondary filter are present. Primary filter: -absorbs visible radiation and transmit UV radiation. Secondary filter :-absorbs UV radiation and transmit visible radiation.

Thin-Film Filters   :

Thin-Film Filters   Thin-film filters are also available to specifically transmit or reject laser lines . Laser light can contain additional wavelengths in addition to the main laser line. This emission is referred to as the plasma emission, which typically occurs over a range of wavelengths and is not strongly directional.

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Monochromators: they convert polychromatic light into monochromatic light . They can isolate a specific range of wavelength or a particular wavelength of radiation from a source.   Excitation monochromators :- provides suitable radiation for excitation of molecule . Emission monochromators :- isolate only the radiation emitted by the fluorescent molecules.

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SAMPLE HOLDERS The majority of fluorescence assays are carried out in solution. Cylindrical or rectangular cells fabricated of silica or glass used. Path length is usually 10mm or 1cm . All the surfaces of the sample holder are polished in fluorimetry . Square cuvettes or cells will be found to be most precise since the parameters of pathlength and parallelism are easier to maintain during manufacture.

EFFECTS OF SAMPLE GEOMETRY:

EFFECTS OF SAMPLE GEOMETRY The apparent fluorescence intensity and spectral distribution can be dependent upon the optical density of the sample, and the precise geometry of sample illumination. The most common geometry used for fluorescence is right angle observation of the center of a centrally illuminated cuvette(top left). Other geometric arrangements include Front-face and Off-center illumination.

SAMPLE GEOMETRY:

SAMPLE GEOMETRY

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Off center illumination decreases the path length, which can also be accomplished by using cuvettes with path lengths less than 1 cm . These methods are generally used to decrease the inner filtering effects due to high optical densities or to sample turbidity. front-face illumination is performed using either triangular cuvettes or square cuvettes oriented at 30 to 60 degree relative to the incident beam In our opinion, an angle of 45 degree should be discouraged. A large amount of light is reflected directly into the emission monochromator , increasing the chance that stray light will interfere with the measurements .

COMMON ERRORS IN SAMPLE PREPARATION:

COMMON ERRORS IN SAMPLE PREPARATION The sample can be too concentrated , in which case all the light is absorbed at the surface facing the light source With highly absorbing solutions and right-angle observations the signal levels can be very low The sample contains a fluorescent impurity , or the detected light is contaminated by Rayleigh or Raman scatter. Intensity fluctuations can be due to particles that drift through the laser beam, and fluoresce or scatter the incident light. Even if the fluorescence is strong, it is important to consider the possibility of two or more fluorophores , that is, an impure sample .

DETECTORS:

DETECTORS PHOTO MULTIPLIER TUBES CHARGE-COUPLED DETECTORS

PHOTO MULTIPLIER TUBES :

PHOTO MULTIPLIER TUBES These are incorporated in expensive instruments like spectrofluorimeter. Its sensitivity is high due to measuring weak intensity of light. The principle employed in this detector is that, multiplication of photoelectrons by secondary emission of electrons. This is achieved by using a photo cathode and a series of anodes ( Dyanodes ). Up to 10 dyanodes are used. Each dyanode is maintained at 75-100V higher than the preceding one

PHOTO MULTIPLIER TUBES :

PHOTO MULTIPLIER TUBES At each stage, the electron emission is multiplied by a factor of 4 to 5 due to secondary emission of electrons and hence an overall factor of 10 6 is achieved. PMT can detect very weak signals , even 200 times weaker than that could be done using photovoltaic cell. Hence it is useful in fluorescence measurements. PMT should be shielded from stray light in order to have accurate results.

CCD Detectors :

CCD Detectors There is a growing usefulness of charge-coupled devices (CCDs) in fluorescence spectroscopy. CCDs are imaging detectors with remarkable sensitivity and linear dynamic range. CCDs typically contain 106 or more pixels . Each pixel acts as an accumulating detector where charge accumulates in proportion to total light exposure. The charge at each pixel point can be read out when desired, to obtain a two-dimensional image. CCDs are used widely in fluorescence microscopy

CCD Detectors :

CCD Detectors Small spectrofluorometers using CCDs are commercially available.These devices are conveniently interfaced via a USB cable and have no moving parts. When combined with an LED light source the entire instrument becomes a solid state device.  

INSTRUMENTS:

INSTRUMENTS SINGLE BEAM FLUORIMETER DOUBLE BEAM FLUORIMETER SPECTROFLUORIMETER

SINGLE BEAM FLUORIMETER :

SINGLE BEAM FLUORIMETER It contains tungsten lamp as a source of light and has an optical system consists of primary filter. The emitted radiations is measured at 90 0 by using a secondary filter and detector. Primary filter absorbs visible radiation and transmit UV radiation which excites the molecule present in sample cell.

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Single beam instruments are simple in construction cheaper and easy to operate.

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ADVANTAGES Simple in construction Easy to use. Economical DISADVANTAGES It is not possible to use reference solution & sample solution at a time.

DOUBLE BEAM FLUORIMETER :

DOUBLE BEAM FLUORIMETER It is similar to single beam except that the two incident beams from a single light source pass through primary filters separately and fall on the another reference solution. Then the emitted radiations from the sample or reference sample pass separately through secondary filter and produce response combinely on a detector.

DOUBLE BEAM FLUORIMETER :

DOUBLE BEAM FLUORIMETER SPLITTER

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ADVANTAGES Sample & reference solution can be analyzed simultaneously. DISAVANTAGES Rapid scanning is not possible due to use of filters.

SPECTROFLUORIMETER :

SPECTROFLUORIMETER In this primary filter in double beam fluorimeter is replaced by excitation monochromator and the secondary filter is replaced by emission monochromator . Incident beam is split into sample and reference beam by using beam splitter.  

SPECTROFLUORIMETER :

SPECTROFLUORIMETER

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Advantages Rapid scanning to get Excitation & emission spectrum. More sensitive and accuracy when compared to filter fluorimeter .

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